Type-I IFNs are a family of secreted cytokines made by host cells in response to pathogens and whose production is strictly regulated. Type I IFNs control the transcription of a large group of genes important in resisting infection and are also required to activate responses of the innate and adaptive immune systems including antigen presentation and production of cytokines involved in activation of T cells, B cells and natural killer cells. While type I IFN signaling is increasingly appreciated as an important host defense mechanism against invading pathogens, little is known about the bacterial ligands and respective host receptors that trigger type I IFN induction. Our long-term goal is to understand how type I IFN production can be manipulated to prevent and treat auto-inflammatory and infectious diseases. The objective of this proposal, which is our next step in pursuit of that goal is to determine the host factors that contribute to type I IFN production during bacterial infections, using the model cytosolic pathogen Francisella. We hypothesize that multiple type I IFN-stimulating cytosolic receptors are activated by diverse ligands from the cytosolic pathogen Francisella. Recent studies have demonstrated that immune cells express several cytosolic sensors that induce type I IFNs in response to nucleic acid ligands, including DNA, RNA and cyclic-di-nucleotides (CDNs). Thus far, two general mechanisms of activation have been described. First, viable bacteria can secrete stimulatory ligands into the cytosol through specialized secretion systems. Second, ligands can be detected from bacteria that are lysed or degraded. From preliminary experiments, we believe that both mechanisms of activation occur during a Francisella infection. The experiments outlined in this proposal will likely lead to the identification of new factors involved in type I IFN signaling and provide insight on the molecular mechanisms employed by the host to protect against pathogens. We propose to study the mechanisms that lead to type I IFN production during infections with Francisella by pursuing the following three aims. Aim 1: Determine if CDNs are important for activation of type-I IFNs during Francisella infection. Aim 2: Identify Francisella factors that modulate the cytosolic innate immune response. Aim 3: Identify cytosolic sensors that regulate type-I IFN induction in response to Francisella. We will take targeted genetic and unbiased forward genetic screen approaches to identify Francisella factors that modulate type I IFN production. Additionally, we will take genetic and biochemical approaches to identify cytosolic sensors that contribute to type I IFN signaling. This work will increase our understanding of cytosolic detection, innate immunity and host-pathogen interactions.